A distal locus control region (LCR) regulates the human growth hormone (hGH) gene cluster. The determinants of the hGH LCR, located between 14.5 to 32 kb 5'to the hGH-N gene are marked by five DNaseI hypersensitive sites (HSI-HSV). These determinants act in concert to establish robust and appropriately regulated expression of hGH-N in pituitary somatotropes. HSI is the primary determinant of hGH-N transgene expression in somatotropes. HSI contains three essential binding sites for the pituitary-enriched transfactor Pit-1. These HSI core elements appear to function via specific recruitment of histone acetyltransferase activity, leading to the establishment of a 32 kb domain of core histone hyperacetylation that encompasses the entire hGH LCR and extends to the distal hGH-N promoter. The powerful Pit-1 array at HSI exerts unique, chromatin-based functions that are dominant over the activity of the Pit-1 sites at the hGH-N promoter. The full level of HSI activity is dependent on synergism with a closely linked determinant. HSII, a pituitary-specific HS located -1 kb 5'to HSI, may be responsible for this synergism. The terminal boundaries of the 32 kb hGH LCR domain are specific to pituitary cells and in a transgenic setting serve to insulate the hGH locus from site-of-integration effects and variegation of expression. Present data suggest that the boundary and/or insulator activities at the 5'border of this domain may be mediated by HSV. A boundary element located 3'to the hGH-N gene may insulate the placental hCS genes from HSI-mediated activation in the pituitary. The role of the LCR in the initial activation of the hGH locus may be followed by an equally important role in maintenance of epigenetic modifications within the chromatin domain that ensure continued hGH-N expression throughout adult life. The Specific Aims of this proposal focus on these major aspects of hGH LCR function: Aim I. Characterize interactions at HSI that initiate hGH LCR activation, Aim II. Define the mechanism(s) of long-range activation of hGH-N by its LCR, Aim III. Characterize the role of HSII as an HSI facilitator, Aim IV. Identify the chromatin boundaries that establish the pituitary-specific hGH LCR domain, and Aim V. Define the role of HSI in stable maintenance of hGH-N expression. Significantly, these studies will address fundamental epigenetic mechanisms involved in the activity of LCRs, and establish a foundation for understanding normal and pathological aspects of hGH-N gene control.